1. Field of the Invention
The present invention relates to devices and methods for the detection of a bacterial toxin in a biological sample. In particular, the invention relates to a lateral flow assay for identifying the presence of biomolecules produced by Staphylococcus aureus isolates, such as Panton-Valentine Leukocidin (PVL) and PBP2a.
2. Background Information
The following Background Information is intended to aid the reader in understanding the invention and is not admitted to be prior art.
Staphylococcus aureus is a clinically-relevant gram-positive coccus. About 20-30% of a healthy human population carries S. aureus on mucous membranes. S. aureus can cause a wide range of diseases, including sepsis, toxic shock, pneumonia, skin and soft tissue infections, and infection of bones and synthetic implants. S. aureus has also been detected in a wide range of animals.
Methicillin-resistant Staphylococcus aureus (MRSA) is S. aureus that harbor an alternate penicillin-binding protein, known as PBP2a, encoded by the gene mecA and different alleles thereof. As the name implies, MRSA can be detected by the observation of S. aureus growth in presence of methicillin, as well as other beta-lactam antibiotics such as penicillins, cephalosporins and carbapenems.
Because of limited treatment options, MRSA is a significant cause of morbidity and mortality of hospital patients, and poses a challenge to infection control and public health. Due to the need for expensive second-line drugs and quarantine measures, MRSA causes considerable costs to healthcare providers. There are an estimated 53 million MRSA carriers in the world and 2.5 million MRSA carriers in the United States.
The Panton-Valentine leukocidin (PVL) toxin is a phage-borne virulence factor of Staphylococcus aureus. It is a clinically-important phage borne virulence factor in S. aureus and MRSA. PVL is encoded by two adjacent and co-expressed genes, lukS-PV and lukF-PV (lukS-PV, lukF-PV, GenBank BA000033.2:MW1378 and MW1379). A T-cell epitope of lukS-PV capable of eliciting strong proliferation of LST cells has been recently characterized: N169 YISEVERQNSKSVQWGIKANSFIT193 (Brown, et al., Open J. Immunol., 2(3):111-115 (2012)). Polymers of these molecules form pores in human leukocyte membranes leading to cell death and cytokine release. Alternatively, low concentrations may induce apoptosis in granulocytes.
PVL is related to gamma-hemolysin (lukF/S-hlg) and to other leukocidins (lukE/D, lukM/lukF-P83 in S. aureus and lukF/S-int in S. intermedius/pseudintermedius). PVL is structurally, and in terms of sequence similarities, related to other leukocidins, such as lukE/D, lukM/lukF-P83 in S. aureus and lukF/S-int in S. intermedius/pseudintermedius, and to the hlgA/lukF/S-hlg gamma-hemolysin/leukocidin locus.
As discussed above, PVL is toxic for human leukocytes because it forms polymeric pores in the cell membranes of white blood cells. Leukocyte death results in cytokine release and attracts new white blood cells. PVL genes are phage-borne and mobile; they can be found in very diverse clonal complexes (e.g., CC1, 5, 8, 15, 22, 25, 30, 45, 59, 72, 80, 88, 93, 96/154, 121, 188, 398). So far, PVL is restricted to S. aureus strains isolated from humans. S. aureus from ruminants (e.g., cattle, goats and sheep) have another specific leukocidin, encoded by the genes lukM and lukF-P83 (e.g., in CC479, 151, 133, 97, 30, 20).
PVL is frequently detected in S. aureus isolates from skin and soft tissue infections (SSTI) as is associated with chronic/recurrent infections such as furunculosis, especially in young and previously healthy adults. PVL-positive S. aureus can also cause more severe diseases such as necrotizing pneumonia. This condition is occasionally a complication of other respiratory tract infections such as influenza and its fatality rate can be as high as 40%. In contrast, PVL is rarely isolated in S. aureus from healthy carriers or from isolates associated with other types of infections, such as bacteremia.
Although PVL was described in the 1930s, its existence as a potent leukotoxic toxin produced by some S. aureus strains was postulated already in the late 19th century (28). In the 1940s and 1960s, worldwide outbreaks of PVL-positive, methicillin-susceptible S. aureus were observed, and by the late 1990s, PVL-positive community acquired MRSA (caMRSA) had emerged.
Many clinical conditions can be related to PVL, including skin and soft tissue infections, abscesses, furunculosis (boils), and mastitis. These conditions range from minor infections to life-threatening conditions, such as necrotizing fasciitis. PVL-associated infections tend to be chronic or recurring. S. aureus is also an occasional cause of pneumonia, often as a superinfection or a complication of influenza. Necrotizing pneumonia, the most serious form of pneumonia, is commonly associated with PVL, and it is often fatal.
PVL is extremely rare among S. aureus isolates from healthy carriers or from implant-associated infections. PVL is common among isolates from infections such as abscesses or furuncles. Because of the tendency to cause chronic, recurrent or particularly severe infections, PVL-positive S. aureus strains warrant different, more aggressive treatment than “normal” S. aureus strains. In Great Britain, this is already officially recommended by a guideline by the Health Protection Agency.
To date, PVL detection is primarily achieved using a molecular method that is essentially limited to reference centers and specialized laboratories with equipment and experience to perform such assays. Current methods for detecting PVL and PBP2a include polymerase chain reaction (PCR) for the identification of PVL and PBP2a genes. PCR can be performed only in specialized laboratories with dedicated hardware and trained personnel, and requires sample preparation. Patients who present to family physicians and primary care centers may not have ready access to such laboratory facilities. These cases remain undiagnosed and thus possibly not adequately treated, resulting in greater health risks to the patient and potential economic consequences to doctors and hospitals. Other methods for the identification of PVL producing S. aureus and methicillin-resistant S. aureus, such as those disclosed in US 2010/0129839, require pretreatment (i.e., heating) of the biological sample to denature the PVL, in addition to more time and work consuming immunological assays such as ELISA.
Therefore, a continuing need exists for methods and devices for rapid detection of PVL, PBP2a and S. aureus Protein A (SPA) with minimal sample processing, while ensuring accurate and reliable results. A simple, rapid assay could facilitate diagnosis of PVL-associated disease in primary and secondary health care facilities as well as determine whether the strain in methicillin-resistant. Rapid assays save time, as results from reference laboratories often take several days or weeks. A test which distinguishes PVL-positive MRSA from PVL-negative MRSA strains may ultimately result in greater treatment benefits to the patient and assist in preventing the spread of the former within hospital settings. Further, a test which distinguishes PBP2a-positive MRSA from PBP2a-negative MRSA strains may ultimately result in greater treatment benefits to the patient and assist in preventing the spread of the former within hospital settings.